Publications: protein design, synthetic biology

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Protein-based surfaces

Self-Assembling Protein Surfaces for In Situ Capture of Cell-Free-Synthesized Proteins

Ella Lucille Thornton, Sarah Maria Paterson, Zoe Gidden, Mathew H. Horrocks, Nadanai Laohakunakorn and Lynne Regan

Frontiers in Bioengineering and Biotechnology 2022 10:915035 

In this paper we show a new method for capturing cell-free expressed proteins onto a surface. BslA is used to form a functionalised monolayer on the surface of a cell-free reaction chamber onto which cell-free expressed (or pure protein) can be covalently captured using “Catcher/Tag” technology. We expect that this surface capture method will have a lot of different applications.

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Facile protein immobilization using engineered suface-active biofilm proteins

Danielle M. Williams, Gilad Kaufman, Hadi Izadi, Abigail E. Gahm, Sarah M. Prophet, Kyle T. Vanderlick, Chinedum O. Osuji and Lynne Regan.

ACS Applied Nano Materials 2018 1:2483–2488

Here we use a combination of natural proteins with unusual physical properties (BslA1, SpyCatcher/SpyTag, SnoopCatcher/SnoopTag) to create surfaces to present and display a variety of proteins. This work is important because for biosensor applications, the ability to present a protein on a surface, whilst simultaneously preventing both its interaction with that surface and the non-specific binding of analytes to the surface, is vital.  

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Modulating the Viscoelastic Properties of Covalently Crosslinked Protein Hydrogels

Rossana Boni, Lynne Regan.

Gels 2023 9(6), 481

We have successfully designed and programmed the SpyTag (ST) peptide and SpyCatcher (SC) protein, that spontaneously form covalent crosslinks upon mixing, to form hydrogels with defined physical characteristics. We demonstrated how differences in the composition of the microscopic building blocks change the macroscopic viscoelastic properties of the hydrogels. We specifically investigated how the identity of the protein pairs, the molar ratio of ST:SC, and the concentration of the proteins influence the viscoelastic response of the hydrogels. By showing tuneable changes in protein hydrogel rheology, we increased the capabilities of synthetic biology to create novel materials, allowing engineering biology to interface with soft matter, tissue engineering, and material science.

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Chemically cross-linked hydrogels from repetitive protein arrays

Rossana Boni, Elizabeth A. Blackburn, Dirk-Jan Kleinjan, Mantas Jonaitis, Flora Hewitt-Harris, Megan Murdoch, Susan Rosser, David C. Hay, Lynne Regan.

Journal of Structural Biology 2023

In this paper we generate hydrogels using the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein which spontaneously form covalent crosslinks upon mixing. We found that changing the ratios of the protein building blocks (ST:SC) alters the viscoelastic properties and gelation speeds of the hydrogels. We also show that the HepG2 cell line constitutively expressing GFP remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. This demonstrates the potential of engineered protein hydrogels as scaffolds for tissue regeneration. 

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Stimuli-responsive smart gels realized via modular protein design

Tijana Z. Grove, Chinedum O. Osuji, Jason D. Forster, Eric R. Dufresne and Lynne Regan.

ASC Journal of the American Chemical Society 2010 132:14024-6.

Here we use designed repeat protein arrays, in combination with multi-dentate peptide cross-linkers, to make stimuli-responsive hydrogels. These gels form and ‘dissolve’ reversibly in response to specific molecular stimuli. Moreover, their macroscopic viscoelastic properties are specified by the molecular details of their construction. This work is important because the ability to create gel matrices of user-prescribed stiffness is vital for many applications in tissue engineering and stem cell technologies. 

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Proteins as live cell imaging tools

Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide–Peptide Interactions

Zoe Gidden, Curran Oi, Emily J. Johnston, Zuzanna Konieczna, Haresh Bhaskar, Lorena Mendive-Tapia, Fabio de Moliner, Susan J. Rosser, Simon G. J. Mochrie, Marc Vendrell, Mathew H. Horrocks, and Lynne Regan.

ACS Nano Letters 2023

In this paper we use interacting peptides to label proteins that do not tolerate direct fusions to fluorescent proteins with peptide tags as short as 5-residues. By using difficult to label yeast membrane proteins as an example, we show that these short peptide tags do not perturb the normal location or function of the protein target. We also demonstrate that this technique is compatible with both live-cell diffraction-limited and super-resolution microscopy techniques, the latter enabling quantification of protein arrangement at the nanoscale. In addition we successfully use orthogonal peptide interaction pairs to carry out live-cell super-resolution imaging of multiple proteins concurrently. 

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Live-cell super-resolution imaging of actin using LifeAct-14 with a PAINT-based approach

Haresh Bhaskar, Dirk-Jan Kleinjan, Curran Oi, Zoe Gidden, Susan J. Rosser, Mathew H. Horrocks and Lynne Regan.

Protein Science 2022

In this paper we show that labelled binding peptides can be used to carry out super-resolution imaging of protein structures in live cells in an approach called direct-LIVE-PAINT. This technique is similar to the previously published LIVE-PAINT technique from our lab but it does not require a partner peptide tag on the protein-of-interest since the labelled peptide transiently binds directly to the protein being imaged. We use an actin binding peptide fused to EGFP to demonstrate this technique in live mammalian cells and we were able to observe the dynamic nature of actin filaments.

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LIVE-PAINT allows super-resolution microscopy inside living cells using reversible peptide-protein interactions

Curran Oi, Zoe Gidden, Louise Holyoake, Owen Kantelberg, Simon Mochrie, Mathew H. Horrocks and Lynne Regan.

Communications Biology 2020 3(1):1-10.

In this paper we introduce LIVE-PAINT, a novel imaging strategy where reversibly interacting peptide pairs are used to generate super-resolution images of proteins in living cells. One peptide is added to the protein of interest while the other is added to a fluorescent protein that is expressed separately. When the peptides interact, the fluorescent protein is held stationary long enough that a 'blink' is observed and during analysis the exact location of this blink can be determined. Over many blinks a super-resolution image of the protein of interest can be generated. This improves on current live cell super-resolution methods, like PALM, as the tag is small and thus less perturbative to the protein being studied, and LIVE-PAINT can be easily multiplexed through the use of orthogonal peptide pairs. 

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A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking

Hinrichsen M, Lenz M, Edwards JM, Miller OK, Mochrie SGJ, Swain PS, Schwarz-Linek U, and Lynne Regan.

Protein Engineering, Design & Selection 2017 30:771-780.

Here we used a designed protein-peptide pair to covalently label proteins, post-translationally, in live cells. This work is important because labelling a protein when it is in its final, functional, state is less perturbing to its function, as we demonstrate.  We use our method, combined with microfluidics and single cell tracking, to study protein life-times in the plasma membrane of yeast (S. cerevisiae). 


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Designed proteins as novel imaging reagents in living Escherichia coli

Susan E. Pratt, Elizabeth B. Speltz, Simon G. J. Mochrie and Lynne Regan.

Chembiochem 2016 117:652-7.

Here we used designed protein-peptides to non-covalently label proteins in live cells. This work is important because we designed protein-peptide pairs of different affinities, (measured in vitro), and we then demonstrated how those different affinity pairs function in vivo, in live E. coli.

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Designed proteins for modulating cellular networks and building structures

Designed proteins to modulate cellular networks

Aitziber L. Cortajarena, Tina Y. Liu, Mark Hochstrasser and Lynne Regan.

ACS Chemical Biology 2010 5:545-52.

Here we used a combination of design and selection to obtain protein modules that bind to a specific region of a cellular protein (SEM1). This work is important, because we showed that by expressing the protein modules in yeast, we can inhibit the function of SEM1, with clear phenotypic consequences.

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Design of protein-peptide interaction modules for assembling supramolecular structures in vivo and in vitro

Elizabeth B. Speltz, Aparna Nathan and Lynne Regan.

ACS Chemical Biology 2015 10:2108-15.

Here we designed protein modules that bind to short peptides. This work is important because the binding specificities of these modules are orthogonal to each other and to all yeast proteins. Thus, they can be used to assemble supramolecular structure, both in vitro and in vivo.

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